Pharmaceutical Hydrates Analysis—Overview of Methods and ...

pharmaceutics

Review

Pharmaceutical Hydrates Analysis—Overview ofMethods and Recent Advances

Ewa Jurczak 1,†, Anna Helena Mazurek 1,† , Łukasz Szeleszczuk 1,* , Dariusz Maciej Pisklak 1

and Monika Zielinska-Pisklak 2

1 Department of Physical Chemistry, Chair and Department of Physical Pharmacy and Bioanalysis,Faculty of Pharmacy, Medical University of Warsaw, Banacha 1 str., 02-093 Warsaw, Poland;[email protected] (E.J.); [email protected] (A.H.M.); [email protected] (D.M.P.)

2 Department of Biomaterials Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1 str.,02-093 Warsaw, Poland; [email protected]

* Correspondence: [email protected]; Tel.: +48-501-255-121† These authors contributed equally to the work.

Received: 1 September 2020; Accepted: 7 October 2020; Published: 11 October 2020�����������������

Abstract: This review discusses a set of instrumental and computational methods that are used tocharacterize hydrated forms of APIs (active pharmaceutical ingredients). The focus has been put onhighlighting advantages as well as on presenting some limitations of the selected analytical approaches.This has been performed in order to facilitate the choice of an appropriate method depending onthe type of the structural feature that is to be analyzed, that is, degree of hydration, crystal structureand dynamics, and (de)hydration kinetics. The presented techniques include X-ray diffraction(single crystal X-ray diffraction (SCXRD), powder X-ray diffraction (PXRD)), spectroscopic (solidstate nuclear magnetic resonance spectroscopy (ssNMR), Fourier-transformed infrared spectroscopy(FT-IR), Raman spectroscopy), thermal (differential scanning calorimetry (DSC), thermogravimetricanalysis (TGA)), gravimetric (dynamic vapour sorption (DVS)), and computational (molecularmechanics (MM), Quantum Mechanics (QM), molecular dynamics (MD)) methods. Further, thesuccessful applications of the presented methods in the studies of hydrated APIs as well as studies onthe excipients’ influence on these processes have been described in many examples.

Keywords: hydrates; anhydrous; hydration; dehydration

1. Introduction

Polymorphism is a phenomenon defined as the possibility of one chemical substance to exist inseveral different crystallographic forms [1] depending on the temperature, pressure, and humidity aswell as solvents applied during the crystallization process. Polymorphic forms of active pharmaceuticalingredients (APIs) may differ in certain important properties, such as solubility in water, dissolutionrate, melting point, stability, tabletability, and others, which consequently can have an influenceon the drug stability and bioavailability [1]. Different group of structures, although in somerespects similar to polymorphs, are hydrates. They form a subtype of solid solvates in whichwater molecules are incorporated into the crystal lattice of a compound [2]. In comparison withrelated anhydrates, they exhibit a different structure, as it may be altered by the complex H-bondingnetwork [3]. As a consequence, they may also have different physical and chemical properties thantheir anhydrous counterparts. These parallels to polymorphs are the reasons hydrates were previouslycalled pseudo-polymorphs [4,5]. This naming can be found in old manuscripts, however, nowadays it isno more accepted as the correct one in relation to hydrates. The topic becomes even more complicatedwhen a particular hydrate in itself has different polymorphic forms, for example, nitrofurantoin

Pharmaceutics 2020, 12, 959; doi:10.3390/pharmaceutics12100959 www.mdpi.com/journal/pharmaceutics

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monohydrate or niclosamide monohydrate [6]. In such cases, the stoichiometry of the system, thenumber of both host molecules and water molecules, is preserved and only changes in the arrangementof the constituents in the crystal lattice, and alterations in unit cell parameters occur as well.

Hydrates are of particular interest among solid APIs solvates for several reasons. First, the uniquecharacter of the water molecule—its relatively small size and the possibility to form the interactions asboth a donor and acceptor of H-bonding, sometimes simultaneously, make it an important “buildingmaterial” in the field of crystal engineering. Further, from the pharmaceutical point of view, it is anon-toxic substance, in contrast to most of the other organic solvents. Finally, owing to the present ofthe moisture in the air, spontaneous hydration may occur at any stage of drug production or storage,leading to hydrate formation.

Considering the structure, the most common are layer, void, and channel hydrates (Figure 1).They can be also divided into stoichiometric and non-stoichiometric ones. Stoichiometric hydratesare composed of a constant number of water molecules located in clearly defined structure elementslike channels [7]. On the contrary, non-stoichiometric hydrates have a variable number of watermolecules incorporated in a crystal lattice and, in this case, their naming, for example, dihydrate,usually indicates the maximum number of H2O molecules present in a structure. Non-stoichiometrichydrates can occur as channel or void hydrates, but the solvent molecules are disordered because of theweaker H-bonds with the host molecules, when compared with stoichiometric hydrates [8]. As a result,water diffusion out of these structures is much easier. Furthermore, their hydration level dependshighly on the humidity in the surrounding atmosphere. Moreover, non-stoichiometric hydrates aremuch less prone to collapse after water removal. This phenomenon is characteristic for stoichiometrichydrates, especially when they form large channels or voids. In such cases, distortion of a complexH-bonding network that was stabilizing the stoichiometric hydrate leads to completely new moleculararrangements. Less prevalent among API hydrates are ion coordinated hydrates (known also asion-associated hydrates) [9] and isolated site hydrates. In case of the latter, water molecules interactsolely with the host molecule, but not with each other [10].

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(a)

(b)

(c)

Figure 1. Examples of different types of hydrates: (a) isolated hydrate, dapsone; (b) channel hydrate, brucine [11]; and (c) ion-coordinated hydrate, ondansetron hydrochloride [12]. Crystallographic structures were downloaded from the Cambridge Crystallography Data Centre (CCDC) [13] with the following reference codes: ANSFON (dapsone hydrate), YOYZIX (brucine hydrate), and YILGAB (ondansetron hydrochloride dihydrate). Ondansetron hydrochloride: chlorine is marked in green.

Figure 1. Examples of different types of hydrates: (a) isolated hydrate, dapsone; (b) channel hydrate,brucine [11]; and (c) ion-coordinated hydrate, ondansetron hydrochloride [12]. Crystallographicstructures were downloaded from the Cambridge Crystallography Data Centre (CCDC) [13] with thefollowing reference codes: ANSFON (dapsone hydrate), YOYZIX (brucine hydrate), and YILGAB(ondansetron hydrochloride dihydrate). Ondansetron hydrochloride: chlorine is marked in green.

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Statistically, approximately one-third of pharmaceutical solids exist in at least two forms, differingin the level of hydration [14]. In most cases, in comparison with anhydrous forms, hydrates arethermodynamically more stable under normal conditions. As a result, they are less prone to dissolve inwater and, consequently, they usually exhibit lower bioavailability, which is an obvious disadvantagein terms of their therapeutic applications [15]. This key property can be improved by the formationof hydrate co-crystals [16] or application of selected excipients [17,18]. On the other hand, hydratesshow better compressibility and tabletability than anhydrates, are less affected by wet granulationprocess, and are less susceptible for the tablet storage conditions like temperature and relative humidity(RH). In such cases, it could be reasonable to find and produce the most stable API hydrate to preventthe form alteration during production or storage. The choice between a hydrated and anhydrousform of an API is very often a critical factor for solid dosage forms’ stability and their mechanicalbehavior [19–21].

Nevertheless, there are some exceptions from the above given principles. For example,erythromycin dihydrate shows a more efficient dissolution rate than the respective monohydrate andanhydrate [22,23]. Theophylline monohydrate exhibits better solubility in water than its anhydrousform [24]. These cases indicate the variability in properties of hydrated APIs and necessity to performcomparative studies between the differently hydrated forms in each particular case.

While, from the legal point of view in most countries, for each polymorphic form of an API,a separate patent may be assigned, the situation is more complicated for hydrates. More specifically,in some countries, the given two forms of API differing in hydration level can be perceived as legally thesame forms, while this is not the case in others. For example, varying in hydration levels, different formsof cefdinir have received individual patent claims in the United States [25]. By contrast, however, theBrazilian and Argentine Patent Law Guidelines exclude the possibility to patent solvates or hydrates,because they both consider them to be discoveries and not new inventions [26,27]. Nonexistence of aworldwide coherent law on this topic has already brought court trials.

Equally disordered is hydrates’ representation in pharmacopoeias, as some of the hydrates areincluded in separate monographs and some are not, without any clear justification. In pharmacopoeias,one can find only those hydrated forms of drugs that are most commonly used, as well as hydratesof widely applied excipients like lactose monohydrate [28]. For example, in the InternationalPharmacopeia [29], one can find separate monographs for both caffeine anhydrate and caffeinemonohydrate. However, in the case of carbamazepine, a monograph for the anhydrous form only canbe found, even though carbamazepine dihydrate is a well-known and commonly applied form of thisAPI [30].

The above shortly enumerated various issues regarding APIs’ hydrates indicate the complexity ofthe discussed subject, which can also be a real challenge for the pharmaceutical industry. However,when properly employed, the ability of most of the APIs to form solid hydrates can be also a uniqueopportunity to improve their stability, processability, or biopharmaceutical properties or even to patenta new solid state form, similarly as in the case of polymorphs. However, for these purposes, a detailedanalysis of hydrated pharmaceuticals is essential.

This review aims to compare the analytical methods, both instrumental and computational, usuallyapplied in API hydration studies. A particular emphasis is put on their strengths and limitations,supported by the references to the recently published works in which they were successfully applied.The intention of the authors was not to describe in a very detailed way each of the analytical methods,but rather to show their applications and present their capabilities in the analysis of APIs hydrates.As most of the methods described in this work can be used to study both the structure of hydrates andtheir stability, as well as the hydration and dehydration processes, firstly, selected analytical techniquesare introduced and, subsequently, their reported applications are summarized. The aim of this studywas, besides presenting the recent advances in this topic, to facilitate the choice of the proper analyticalmethod when exploring the APIs’ hydrates.

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2. Methods Applied for Analysis of Hydrates

2.1. Structure Determination

A starting point in the organic solids analysis is usually detailed determination of their compositionand structure. It is especially interesting and important in terms of hydrates, because often, not onlypolymorphic forms transition, but also change of the hydration degree may occur when the sample isexposed to various temperature, pressure, or humidity conditions.

The difficulty of studies dealing with pharmaceutical hydrates lies in the fact that two different typesof molecules are present in a crystal structure, namely API and water. This triggers a stepwise analysis.Firstly, an insight into the number of water molecules per the host substance molecule is needed. This isconsistent with contradistinction between stoichiometric and non-stoichiometric hydrates. For thispurpose, thermo-analytical method TGA (thermogravimetric analysis) and gravimetric method DVS(dynamic vapour sorption) are usually used. Together with another thermo-analytical method DSC(differential scanning calorimetry), they allow detection of mass gain or loss (due to change of the watercontent) [31], determination of enantiotropic/monotropic relationship between polymorphs [32], as wellas melting or crystallization temperatures [33,34]. Usually, DSC and TGA require a smaller amount ofsample (3–10 mg) than DVS (10–30 mg). However, those three methods have some drawbacks andprobably the most significant one is the destruction of a sample during the analysis.

It should be also taken into account that these methods not always can be applied for investigationof a mixture of compounds, for example, preformulates or whole drug products in which, besides APIs,excipients are also present. This always depends on a particular example taken into consideration anda type of physicochemical property that aims to be determined.

When a hydration stoichiometry is known, more precise insight into the crystal structure isprovided by applying SCXRD (single crystal X ray diffraction), PXRD (powder X ray diffraction),ssNMR (solid state nuclear magnetic resonance spectroscopy), FT-IR (Fourier-transformed infraredspectroscopy), and Raman spectroscopy techniques, as well as computational approaches. They canuniquely identify the crystallinity of API and they are used to determine the exact structure of ahydrated crystal, including the positions and dynamics of water molecules. In the last stage, theanalysis of (de)hydration process is sometimes studied. In order to perform it properly, both theknowledge of the structure and results of thermogravimetric approaches are essential.

2.1.1. Structure Determination Techniques

In order to determine the structure of a hydrate, single crystal X-ray diffraction (SCXRD) is usuallyused. It is the most informative, especially when determining the accurate atoms positions, but at thesame time, it is a demanding method. This is because of the usually higher cost of analysis, but moreimportantly, because of the requirement of a stable crystal of usually a minimum of 0.1 mm in size [35].Often, this condition cannot be fulfilled, sometimes solely owing to the nature of the sample, as manyorganic solids do not form stable crystals of the size proper for the SCXRD analysis.

SCXRD is a non-destructive and most willingly used method applied in order to solve a crystalstructure. It should be noticed, however, especially in the analysis of hydrates, that the properdetermination of the hydrogen atoms’ positions may be very difficult for several reasons. First, thehydrogen atom has only one electron, and thus a very low scattering factor. Further, the electrondensity distributions from this one electron around hydrogen atoms are usually displaced or pulledtowards the bonding regions. Another reason may be the dynamics in the crystal lattice in generaland particularly motions involving H atoms that form hydrogen bonds. Finally, the number ofwater molecules, and thus H atoms forming them, may vary or be significantly disordered in thenon-stoichiometric hydrates.

One of the most commonly applied methods in the crystal structure studies is powder X-raydiffraction (PXRD). PXRD delivers data on the unit cell parameters, and thus sometimes serves as analternative route to a single crystal X-ray diffraction (SCXRD) when the latter is not attainable [36].

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Further, in contrast to SCXRD, PXRD can be applied to study the mixture of solids and, in some cases,determine the quantitative phase composition [37]. More specifically, in terms of hydrate investigation,PXRD helps to differentiate between the hydrated and dehydrated form of the substance. This isplainly visible as a drastic change of a pattern on the diffractogram [38] (Figure 2).

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cases, determine the quantitative phase composition [37]. More specifically, in terms of hydrate investigation, PXRD helps to differentiate between the hydrated and dehydrated form of the substance. This is plainly visible as a drastic change of a pattern on the diffractogram [38] (Figure 2).

Figure 2. Comparison of powder X-ray diffraction (PXRD) diffractograms of 17-β-estradiol anhydrate (red) received by heating the 17-β-estradiol hemihydrate (green). Source: author’s archive, more details in [38].

However, PXRD requires complicated interpretation and demanding refinement in order to provide such detailed information on the crystal structure as SCXRD, especially in terms of the atoms’ positions in the unit cell. Nevertheless, applying synchronically different techniques, with an exclusion of highly demanding SCXRD, it has already been successfully used to determine the structure of new crystalline compounds, including APIs’ hydrates [39–41]. What is more, PXRD patterns can be quickly calculated based on the crystal structure files with a high accuracy using specialized software (Reflex) [42]. Such an approach can be used to refine the experimentally obtained data or to differentiate between various polymorphic forms, including hydrates.

While diffraction-based analytical methods, both PXRD and SCXRD, can be used in the cases of crystalline systems, the next method, solid state NMR, can be used to study both crystalline and amorphous materials (Figure 3).

Solid state NMR (ssNMR) represents a totally different approach than the diffraction methods. It is a non-destructive technique that allows qualitative and in some cases also quantitative measurements. However, ssNMR analysis is relatively expensive and has a long data acquisition time [43]. Intuitively, when applying ssNMR to the study of pharmaceutical hydrates, one could think of registering the 1H or 17O spectra. However, those kinds of experiments are rarely performed because of the broad overlapping signals in the solid state 1H NMR spectra and quadrupolar character of the 17O nucleus. A very promising way to overcome the problem of broad signals in the solid state 1H NMR spectra is ultra-fast magic angle spinning (MAS). For example, this approach has recently been successfully used to differentiate the anhydrous and monohydrate form of theophylline [44]. The most commonly performed ssNMR analysis experiments are 13C and 15N, combined with MAS and cross-polarization (CP). In the cases of organic solids, those approaches can be used to distinguish between the forms at different hydration levels (Figure 3), though such experiments do not provide any direct information on water within the crystal structure. Moreover, especially to study the hydration and dehydration processes, 2H NMR experiments are being performed using D2O in the crystallization process or exposing hygroscopic samples in the contact with the vapor of D2O [45]. However, while this approach can be used to investigate the hydrogen bond dynamics in a crystal lattice, it should be noticed that, because of the isotope effect, both the structure and kinetics of hydration/dehydration may be different when D2O is used instead of H2O.

10 12 14 16 18 20 22 24 26 28 30

2Θ [o]

Figure 2. Comparison of powder X-ray diffraction (PXRD) diffractograms of 17-β-estradiol anhydrate(red) received by heating the 17-β-estradiol hemihydrate (green). Source: author’s archive, more detailsin [38].

However, PXRD requires complicated interpretation and demanding refinement in order toprovide such detailed information on the crystal structure as SCXRD, especially in terms of the atoms’positions in the unit cell. Nevertheless, applying synchronically different techniques, with an exclusionof highly demanding SCXRD, it has already been successfully used to determine the structure ofnew crystalline compounds, including APIs’ hydrates [39–41]. What is more, PXRD patterns can bequickly calculated based on the crystal structure files with a high accuracy using specialized software(Reflex) [42]. Such an approach can be used to refine the experimentally obtained data or to differentiatebetween various polymorphic forms, including hydrates.

While diffraction-based analytical methods, both PXRD and SCXRD, can be used in the casesof crystalline systems, the next method, solid state NMR, can be used to study both crystalline andamorphous materials (Figure 3).

Solid state NMR (ssNMR) represents a totally different approach than the diffraction methods.It is a non-destructive technique that allows qualitative and in some cases also quantitativemeasurements. However, ssNMR analysis is relatively expensive and has a long data acquisitiontime [43]. Intuitively, when applying ssNMR to the study of pharmaceutical hydrates, one couldthink of registering the 1H or 17O spectra. However, those kinds of experiments are rarely performedbecause of the broad overlapping signals in the solid state 1H NMR spectra and quadrupolar characterof the 17O nucleus. A very promising way to overcome the problem of broad signals in the solid state1H NMR spectra is ultra-fast magic angle spinning (MAS). For example, this approach has recentlybeen successfully used to differentiate the anhydrous and monohydrate form of theophylline [44].The most commonly performed ssNMR analysis experiments are 13C and 15N, combined with MASand cross-polarization (CP). In the cases of organic solids, those approaches can be used to distinguishbetween the forms at different hydration levels (Figure 3), though such experiments do not provide anydirect information on water within the crystal structure. Moreover, especially to study the hydrationand dehydration processes, 2H NMR experiments are being performed using D2O in the crystallization

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process or exposing hygroscopic samples in the contact with the vapor of D2O [45]. However, whilethis approach can be used to investigate the hydrogen bond dynamics in a crystal lattice, it should benoticed that, because of the isotope effect, both the structure and kinetics of hydration/dehydrationmay be different when D2O is used instead of H2O.Pharmaceutics 2020, 12, x 7 of 25

Figure 3. Comparison of 13C cross-polarization (CP) magic angle spinning (MAS) solid state nuclear magnetic resonance spectroscopy (ssNMR) spectra of diosmin monohydrate (red, 1) and anhydrous diosmin (blue, 2). Source: author’s archive, more details in [46].

ssNMR measurements are often combined with precise quantum chemistry calculations, which facilitates or even enables proper assignment of NMR signals [47]. Application of DFT (density functional theory)-based GIAO [48] or GIPAW [49] methods makes it possible to theoretically calculate the NMR parameters, such as chemical shielding constants and anisotropy tensors. Thus, it enables validation or refinement of the suggested crystal structure. The high accuracy of such an approach has been already acknowledged multiple times [50–52]. For example, calculations of NMR shielding constants in order to simulate the NMR spectra using available crystal structures enable rapid confirmation of whether the analyzed crystal form of a compound is a hydrate or anhydrous one, without the need for any external standards (Figure 4). Nowadays, such a combination of ssNMR and computational methods is so widely used that such an approach has already been defined as ‘NMR crystallography’ [53].

Figure 3. Comparison of 13C cross-polarization (CP) magic angle spinning (MAS) solid state nuclearmagnetic resonance spectroscopy (ssNMR) spectra of diosmin monohydrate (red, 1) and anhydrousdiosmin (blue, 2). Source: author’s archive, more details in [46].

ssNMR measurements are often combined with precise quantum chemistry calculations, whichfacilitates or even enables proper assignment of NMR signals [47]. Application of DFT (densityfunctional theory)-based GIAO [48] or GIPAW [49] methods makes it possible to theoretically calculatethe NMR parameters, such as chemical shielding constants and anisotropy tensors. Thus, it enablesvalidation or refinement of the suggested crystal structure. The high accuracy of such an approachhas been already acknowledged multiple times [50–52]. For example, calculations of NMR shieldingconstants in order to simulate the NMR spectra using available crystal structures enable rapidconfirmation of whether the analyzed crystal form of a compound is a hydrate or anhydrous one,without the need for any external standards (Figure 4). Nowadays, such a combination of ssNMR andcomputational methods is so widely used that such an approach has already been defined as ‘NMRcrystallography’ [53].

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Figure 4. 13C NMR spectra: experimental of anhydrous elagic acid (black) and simulated using CASTEP GIPAW density functional theory (DFT) calculations results for anhydrous elagic acid (blue) and elagic acid dehydrate (red). Source: author’s archive, more details in [54].

Apart from PXRD and NMR described above, probably the quickest and easiest methods used to investigate hydrates are FT-IR and Raman spectroscopies, which are complementary to each other. The presence of the unique bands makes it possible to doubtlessly recognize a crystal substance [55]. These two methods are of a special utility in research performed on hydrates because the presence of water can be recognized owing to signals from the hydroxyl group. The representative stretching band of the hydroxyl group appears at a frequency above 3000 cm-1 [56]. Another interesting aspect is investigation of a hydrogen bonds’ net created as a result of the presence of -OH groups. Often, a relationship between the peak position of such an H-bond and its strength can be defined. Typically, the higher the wavenumber of the O-H peak (even about 3500 cm-1 [57]), the less strong the bond between water and a host molecule in the given hydrate. This, in turn, delivers a suggestion on the overall hydrate structure, because, depending on the character of a host substance, either channel or void hydrate is better stabilized. Consequently, FT-IR and Raman spectroscopies enable analysis of changes in hydrogen bonding that occur during hydration/dehydration process. A good example is rearrangement of this network when estradiol

Figure 4. 13C NMR spectra: experimental of anhydrous elagic acid (black) and simulated usingCASTEP GIPAW density functional theory (DFT) calculations results for anhydrous elagic acid (blue)and elagic acid dehydrate (red). Source: author’s archive, more details in [54].

Apart from PXRD and NMR described above, probably the quickest and easiest methods used toinvestigate hydrates are FT-IR and Raman spectroscopies, which are complementary to each other.The presence of the unique bands makes it possible to doubtlessly recognize a crystal substance [55].These two methods are of a special utility in research performed on hydrates because the presenceof water can be recognized owing to signals from the hydroxyl group. The representative stretchingband of the hydroxyl group appears at a frequency above 3000 cm-1 [56]. Another interesting aspectis investigation of a hydrogen bonds’ net created as a result of the presence of -OH groups. Often, arelationship between the peak position of such an H-bond and its strength can be defined. Typically,the higher the wavenumber of the O-H peak (even about 3500 cm-1 [57]), the less strong the bondbetween water and a host molecule in the given hydrate. This, in turn, delivers a suggestion on theoverall hydrate structure, because, depending on the character of a host substance, either channel orvoid hydrate is better stabilized. Consequently, FT-IR and Raman spectroscopies enable analysis of

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changes in hydrogen bonding that occur during hydration/dehydration process. A good example isrearrangement of this network when estradiol hemihydrate undergoes dehydration (Figure 5) [58].Moreover, these methods can be applied to identify different polymorphic crystal forms amonghydrates. It is based on a careful analysis of the fingerprint spectral region.

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hemihydrate undergoes dehydration (Figure 5) [58]. Moreover, these methods can be applied to identify different polymorphic crystal forms among hydrates. It is based on a careful analysis of the fingerprint spectral region.

Figure 5. Comparison of the Fourier-transformed infrared spectroscopy (FT-IR) spectra of anhydrous estradiol (black) and estradiol hemihydrate (red). Source: author’s archive, more details in [38].

However, when solely applying FT-IR or Raman spectroscopy, it is not possible to determinate a completely unknown structure. Moreover, especially in the FT-IR transmission technique, because of the longer preparation process, a risk is posed by the environmental humidity, which could alter the analysis [59]. This is why, for the highly absorbing probes, ATR-IR (attenuated total reflectance) is used as it requires no sample preparation and is also a non-destructive method [60].

A combination of the above mentioned techniques helps to differentiate between various types of hydrates, mainly between channel and isolated site hydrates. In case of the first ones, FT-IR shows sharp OH-bands at relatively low frequencies, while in TGA, rather wide weight loss ranges are observed, and DSC presents broad endothermic peaks. On the contrary, ion isolated hydrates are characterized by sharp dehydration endotherms in DSC and narrow weight loss ranges in TGA [61,62].

A completely different approach to determine the structure of small organic molecules is presented by the new method: cryo-electron microscopy/microcrystal electron diffraction CryoEM/MicroED [63]. It enables collecting the high-quality MicroED data from nanocrystals, which results in atomic resolution (<1 Å) crystal structures. CryoEM/MicroED requires a small amount of sample, very simple preparation, and can be conducted in a few minutes. One of its most important advantages is the fact that it can be applied to derive a crystal structure from seemingly amorphous powders.

2.1.2. Purely Computational Structure Determination Techniques

Crystal structure prediction (CSP) is an already widely accepted multi-step methodology used to computationally determine the structure of a crystalline material [64–67]. In the field of hydrate studies, it can be used to predict the crystal structure of a hydrate of known stoichiometry. At first, molecular mechanics (MM) is used in order to generate and rank possible compound conformations. Afterwards, selected conformers are subjected to quantum mechanics calculations. The former are performed usually on single molecules, and the latter on whole crystal structures. This enables observation of conformational polymorphism [68] in the first case and packing polymorphism [68] in the second. The (calculations performed at temperature 0 K) lattice energies derived in this process could be qualified if kinetics factors (like temperature) were included. For that purpose, time- and computational power-consuming molecular dynamics (MD) must be applied [69].

0

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0.6

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Figure 5. Comparison of the Fourier-transformed infrared spectroscopy (FT-IR) spectra of anhydrousestradiol (black) and estradiol hemihydrate (red). Source: author’s archive, more details in [38].

However, when solely applying FT-IR or Raman spectroscopy, it is not possible to determinate acompletely unknown structure. Moreover, especially in the FT-IR transmission technique, because ofthe longer preparation process, a risk is posed by the environmental humidity, which could alter theanalysis [59]. This is why, for the highly absorbing probes, ATR-IR (attenuated total reflectance) isused as it requires no sample preparation and is also a non-destructive method [60].

A combination of the above mentioned techniques helps to differentiate between various types ofhydrates, mainly between channel and isolated site hydrates. In case of the first ones, FT-IR showssharp OH-bands at relatively low frequencies, while in TGA, rather wide weight loss ranges areobserved, and DSC presents broad endothermic peaks. On the contrary, ion isolated hydrates arecharacterized by sharp dehydration endotherms in DSC and narrow weight loss ranges in TGA [61,62].

A completely different approach to determine the structure of small organic molecules is presentedby the new method: cryo-electron microscopy/microcrystal electron diffraction CryoEM/MicroED [63].It enables collecting the high-quality MicroED data from nanocrystals, which results in atomic resolution(<1 Å) crystal structures. CryoEM/MicroED requires a small amount of sample, very simple preparation,and can be conducted in a few minutes. One of its most important advantages is the fact that it can beapplied to derive a crystal structure from seemingly amorphous powders.

2.1.2. Purely Computational Structure Determination Techniques

Crystal structure prediction (CSP) is an already widely accepted multi-step methodology used tocomputationally determine the structure of a crystalline material [64–67]. In the field of hydrate studies,it can be used to predict the crystal structure of a hydrate of known stoichiometry. At first, molecularmechanics (MM) is used in order to generate and rank possible compound conformations. Afterwards,selected conformers are subjected to quantum mechanics calculations. The former are performedusually on single molecules, and the latter on whole crystal structures. This enables observation ofconformational polymorphism [68] in the first case and packing polymorphism [68] in the second.The (calculations performed at temperature 0 K) lattice energies derived in this process could bequalified if kinetics factors (like temperature) were included. For that purpose, time- and computational

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power-consuming molecular dynamics (MD) must be applied [69]. Including the entropy input is ofa high importance because it means that the zero-point energy (ZPE) will be calculated. It has beenproven that the ZPE contributes the most to the free energy differences between hypothetical similarstructures [70]. Consequently, its neglection leads to the overestimation of the number of structuresgenerated [64,71]. Furthermore, neglection of the entropy contribution prevents the investigation ofthe enantiotropically-related polymorphs [72].

Lattice energy is calculated on the basis of the inter- and intra-molecular forces. For example,in the Polymorph Predictor software [73], which is one of the packages used for performing CSP, atwo-step procedure is implemented. Firstly, various molecular conformations are generated, andafterwards, optimal hydrogen positions or chemical groups orientations are defined. This processdelivers both inter- and intra-molecular energies of the investigated structure, which together formlattice energy.

The target of CSP application in the studies of hydrates is the same as in the case of other molecules;that is, it helps to elucidate the structural information obtained from the experiment, and proposesa variety of structural models that could possibly be the new thermodynamic stable hydrates orpolymorphs of the existing hydrates [74].

CSP is usually used when there is no structural information on the investigated molecule available.Its results direct the future experimental approach or support the previous assumptions on the existenceand/or structure of the hydrate, as has happened in the case of orotic acid crystal forms [75]. Applicationof CSP enabled to characterize the structural disorder, and showed stacking faults as well as smallvariability in packing indices of the anhydrous orotic acid. Another purpose to apply CSP is a situationwherein the experimental attempts to obtain a proper crystal for the SCXRD measurements have failed.In such a case, CSP delivers viable structures that can serve as an important point of reference for furtherinvestigations performed with techniques other than SCXRD [76]. A good example is usage of the CSP-and NMR-based approach for analysis of catechin methanol hemisolvate-monohydrate [77]. In thisparticular study, an important feature of the computational CSP techniques has been utilized. Namely,the possibility to introduce manual changes into the analyzed crystalline systems. This allows toinvestigate what is not possible regarding hemihydrates in the automatic procedure of calculations [78].

2.2. Kinetics of (De)Hydration Process

The description of a (de)hydration process is mostly based on both experimental and calculationmethods. The former encompasses spectroscopic and thermogravimetric methods as well as microscopyand other techniques like Karl–Fisher titration (K–F titration) [79]. This titration enables to determinewhether water is adsorbed or incorporated in a crystal structure, the latter meaning that the investigatedsample forms a hydrate. It is known in three variants: volumetric, coulometric, and oven coulometricK–F titration [80]. In all cases, the used iodine is proportional to the amount of water, which is calculatedon the basis of the titrator consumed during the titration process. To perform oven coulometric K–Ftitration, the sample is pre-weighted and heated. It is the water vapor that is used in the titration [81].In this last example, thermogravimetric methods are used in order to investigate the thermal stabilityof a probe and determine the minimal temperature needed for the oven coulometric K–F approach.In general, K–F titration is a very sensitive method. It is found in the pharmacopeial monographs ofhydrates, for example, terpin hydrate [82].

If, during the examination of the process with the application of the thermogravimetric methods,big hysteresis in dehydration is observed, it may suggest that the obtained anhydrate is a stable one andit can be analyzed in detail separately by TGA, PXRD, FT-IR, and so on. However, when dehydration isdefined as reversible, application of combination of different methods, including Karl–Fischer titration,is recommended [20].

For proper (de)hydration research, an insight into H-bonding structure is needed. Apart fromthe already mentioned ssNMR and ‘NMR crystallography’, purely computational approaches arealso undertaken. For that purpose, DFT calculations involving application of hybrid functionals

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(most commonly: B3LYP, M05) and polarizable function (an extended basis set like 6-31++G(d,p)or 6-311+G(2d,2p)) are performed [83–87]. Especially, the latter plays a crucial role, as polarizablefunctions are used to properly define the H-bonding. This helps not only to calculate the energy of theintermolecular interactions (performed also in CSP approach), but also, in further steps, to visualizethe H-bonding pattern. Often, it facilitates explanation of the observed (de)hydration mechanism.

In a computational investigation of the (de)hydration kinetics, an important role is also played bycalculation of the solvent-accessible volume, which significantly differs for non-stoichiometric andstoichiometric hydrates [88]. Resulting from water loss, possible collapse of a void or conformationaldisorientation has a direct impact on the above discussed H-bonds network.

2.3. Stability Determination in the Industrial Production

The analysis of (de)hydration kinetics is performed to describe the reaction process and to determinewhether a transformation between the differently hydrated forms under the given conditions wouldtake place spontaneously, and if so, at what rate [89,90]. The decisive parameters are the energy barrierof water diffusion into the lattice as well as the energy of conformational change [85]. Conversely,the thermodynamic stability investigation is based solely on the reactants of the given process.Described as a function of the Gibbs energy change (Gibbs enthalpy, ∆G) [91], for pharmaceuticalpurposes, thermodynamic stability is calculated in isothermal-isobaric conditions (NPT ensemble) [92].Along with molecular dynamics (MD) or compound-water binding energy calculations [93], it candetermine the temperature and pressure values under which the global minimum could be achieved.For the pharmaceutical industry, these data are of crucial importance and are equivalent with occurrenceof the polymorphic phase transition or desolvation, which could appear during product manufacturingor its storage. These calculations enable to establish a stability order among very often numerous(de)hydrated and amorphous forms of API.

Often, calculations are performed with software based on DFT-D (dispersion correction). Such anapproach is already known for high accuracy [94]. However, it must be emphasized that computingthe energy difference between hydrate and anhydrate is more challenging than computation of thealready relatively well-described polymorph transitions [86,95]. The reason is that, most commonlyapplied and so far probably most accurate for this purpose, PBE functional overestimates the energy ofa transition from hydrate to anhydrate [86,96].

In order to experimentally determine the substance stability, measurement of relative humidity(RH) and water activity aw is applied. As aw informs about the substance solubility [97], its value hasa connection with hydrates’ stability order. According to the ICH Guidance for Industry: Q1A(R2)Stability Testing of New Drug Substances and Products, stability measurements for non-sterile productsduring long-term storage should be performed at specified conditions, which are as follows: 25 ◦C/60%RH and aw < 0.60. This is because RH = 60% and aw = 0.60 are established as border values formicroorganisms’ growth [98]. Regular and accurate testing in the topic is of great importance as onlya proper adjustment of RH, aw and temperature parameters allows control of the phase formationprocess and, as a consequence, delivers a stable product [99].

3. Hydrates Structural Analysis, Selected Cases within the Last 10 Years

Observed in the last 10 years, rapid development of both experimental and computational methodsused in structural analysis of solid state substances has its reflection in a number of recently determinedstructures of hydrated APIs [58,100–106]. In the majority of cases, numerous different experimentalmethods (briefly described in Section 2.2) are used within one study in order to properly determine thestructure of a substance [41,77,89,107–109]. A separate group of hydrates form hydrated co-crystals,which are quite common among obtained forms of APIs. Despite being more complex than simplehydrates, their analysis unfolds with application of the same methods [62,110–115].

In case of APIs’ hydrates, the most commonly applied theoretical approaches encompassCSP [86,88,116,117] and NMR crystallography methods [83,118–122], not both of them at once [123,124].

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Because of the common simultaneous application of various analytical methods by the researchers,it is impossible to review the last decade advances in this field method by method. However, it ispossible to divide the published studies with reference to the investigated subjects such as structuredetermination, mechanism of (de)hydratation, stability, and so on.

Determination of a hydrate type is crucial knowledge in order to properly understand theupcoming polymorph or solvate change. In the CSP approach, firstly, water-free high-energy structuresare determined with the aim of calculating the solvent-accessible volume [86]. This helps to directfurther crystal structure prediction for either non-stoichiometric or other well-structured hydrate forms.For example, for a very similar purpose, water interactions with a host molecule in norfloxacin saltshave been put under close investigation [125]. It has been revealed that some of the structures areion-associated hydrates, while the others belong to typical channel hydrates. This structural differencehas a direct impact on the hydrates’ stability as well as their dehydration temperature. Therefore, itdelivers the molecular level explanation for the observed results of thermal studies.

Not only kinetic parameters, but also resulting products of the dehydration reactions are influencedby the hydrate’s type. An example is 5-HT2a and H1 inverse agonist [126], for which two zwitterionichydrates (HyA, Hy2) and three anhydrates (Form I, II, III) are known. HyA is a non-stoichiometrichydrate and has voids clearly separated from each other. On the contrary, Hy2 is a stoichiometricdihydrate with water channels running in a zig-zag manner along the crystallographic b-axis.The consequences of such structural differences are distinct dehydration mechanisms leading todifferent reaction products. Removal of water molecules from HyA results in anhydrate Form I and II,whereas Hy2 dehydration ends with Form III as a product, and it is the only known way to obtainForm III. These types of investigations are of a crucial importance for the pharmaceutical industry.

An in-depth investigation of dapsone hydrates [116] is a good example of a work that involvesa variety of methods and has been performed recently. Application of PXRD allowed to describeH-bonds motifs, while CSP revealed different structural arrangements among forms I, IV, and V.Moreover, new hydrate V was found and an unusual stoichiometry of 0.33-Hy and its isostructuralrelationship with Hydehy was described. What is more, for 0.33-Hy, it has been revealed that watermolecules are located at the isolated sites of a dapsone molecule framework, instead of being situatedin channels [127], as usually happens for such non-stoichiometric hydrates. The amount of gathereddata enabled construction of a very useful (de)hydrogenation chart.

During hydrate examination, a specific accent must be put on the H-bonding investigation. This isbecause these are the H-bonds which are one of the main reasons for the structural alteration of anon-hydrate [128,129], as they are formed not only between water and API molecules, but also inthe form of water bridges [130]. All these aspects contribute to the formation of complex H-bondnetworks. In the case of etoricoxib, a thorough analysis of a newly found hydrate led to a hypothesisthat the reason for a hydrate existence could be a lack of the H-bonding donor groups in the startingstructure [131]. It was only a water molecule that compromised this deficiency and, in the givenconditions, enabled creating a stable etoricoxib form. An analogical example is the case of morphinansfor which hydrates are preferable to anhydrates owing to the presence of H-bonding donors/acceptors,which enables creating a more stable H-bonding network [132].

What is more, H-bonding is also one of the underlying elements for both conformational andpacking polymorphism, also observed among hydrates. The studies targeted on the latter revealwhether mono- or poli-layered systems are formed [87,133] and deliver data on the arrangement ofmolecules within a crystal unit. For example, a vibrational and thermogravimetric analysis of codeinephosphate sesquihydrate [134] led to a conclusion that, depending on the solvation method, differenthydrate polymorphs are obtained and their molecular arrangements are often different from the onespresent in the commercial APIs.

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4. Mechanism of (De)Hydration, Selected Cases within the Last 10 Years

The mechanism of (de)hydration is obviously strongly related to the substance’s structure. Raisedin the last section, the topic of hydrate channels is a very important issue. A good example is thestructure of mildronate dihydrate [135]. Its dehydration process proceeds in a one-step manner,without an intermediate monohydrate, as in most other dehydration cases. It is a consequence of apresence of very large channels, which triggers a loss of whole water at once, instead of proceeding instages. This example plainly shows how structural aspects are linked with the dehydration kinetics andexplains why kinetics of water escape, H-bonding network breakdown, and molecules’ rearrangementwithin a crystal are analyzed simultaneously [84]. It has been shown even more clearly in the exampleof fluconazole [85]. Both structural and kinetic aspects make a hydrated fluconazole form preferable.These are the hydrate structural similarity to anhydrate and low hydration barrier. Unfortunately, theformation of a hydrate lowers drug’s solubility and, as a result, reduces its bioavailability. Such anexample shows that the production of pharmaceuticals prone to hydration forms a real challenge forthe industry. It also indicates that only complex structure-kinetics investigations allow for properunderstanding of the dehydration process and its implications (Figure 6).

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without an intermediate monohydrate, as in most other dehydration cases. It is a consequence of a presence of very large channels, which triggers a loss of whole water at once, instead of proceeding in stages. This example plainly shows how structural aspects are linked with the dehydration kinetics and explains why kinetics of water escape, H-bonding network breakdown, and molecules’ rearrangement within a crystal are analyzed simultaneously [84]. It has been shown even more clearly in the example of fluconazole [85]. Both structural and kinetic aspects make a hydrated fluconazole form preferable. These are the hydrate structural similarity to anhydrate and low hydration barrier. Unfortunately, the formation of a hydrate lowers drug’s solubility and, as a result, reduces its bioavailability. Such an example shows that the production of pharmaceuticals prone to hydration forms a real challenge for the industry. It also indicates that only complex structure-kinetics investigations allow for proper understanding of the dehydration process and its implications (Figure 6).

Figure 6. 13C ssNMR as a method that can be used to study the effect of dehydration temperature on the received anhydrous product form. More information in [38]. Source: author’s archive.

The available methods enable a step by step [136] observation of the changes occurring during dehydration or the water uptake [137]. These studies deliver information that is very important for the pharmaceutical industry. Namely, they can reveal the existence or non-existence of an intermediate in the dehydration process [138], as well as define the kinetics of a conversion of an amorphous substance into a crystalline upon the dissolution [139]. A perfect example of the strong influence of hydration conditions on the received hydrate form is the case of sodium naproxen [140].

Figure 6. 13C ssNMR as a method that can be used to study the effect of dehydration temperature onthe received anhydrous product form. More information in [38]. Source: author’s archive.

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The available methods enable a step by step [136] observation of the changes occurring duringdehydration or the water uptake [137]. These studies deliver information that is very important for thepharmaceutical industry. Namely, they can reveal the existence or non-existence of an intermediatein the dehydration process [138], as well as define the kinetics of a conversion of an amorphoussubstance into a crystalline upon the dissolution [139]. A perfect example of the strong influence ofhydration conditions on the received hydrate form is the case of sodium naproxen [140]. An anhydrousform changes in a one-step process into a dihydrate I, but when the hydration is conducted in twosteps, dihydrate II emerges. Many more far reaching structural consequences can cause dehydrationin a hydrate co-crystal. Water loss from carbamazepine-nicotinamide cocrystal hydrate inducednew co-crystallization between anhydrous carbamazepine and nicotinamide [141], ending up in acompletely new structure. All the changes have been observed thanks to step by step PXRD analysis.

Another crucial aspect for the industry is spontaneous water absorption. In such a process,anhydrate carbamazepine converts into the hydrate form I, whose different crystallinity leads to asignificant decline of the drug’s bioavailability [142]. An adverse process is of no less importance assome APIs undergo water desorption-induced phase transition. Correlation of a weight loss with RHrequires application of, for example, water vapour sorption measurements and Raman spectra, toobtain diagrams of sorption–desorption cycle. This gravimetric-spectroscopy methodology has alreadybeen proposed and effectively applied to determine the optimal storage conditions of drugs [90].

A related topic that is of a huge industrial importance is reversibility of the hydration. Such asituation concerns anhydrate and trihydrate of alendronate monohydrate [116]. Thermogravimetricmeasurements together with computational methods deliver a possible molecular-based explanationfor this phenomenon. The suggested reason is the fact of firstly releasing two non-bonded tothe sodium cation water molecules, and only afterwards proceeding with liberation of the third,cation-solvated water molecule. This plainly shows that solvation of cation is stronger than thestructural interaction of water molecules with the host molecule. Another example described in detail,which is associated with the hydration reversibility topic, is the case of paroxetine hydrochloridehydrates [143]. Here, on the contrary, it has been proven that no sorption–desorption hysteresis can bedepicted due to water presence in channels and not in pores of the hydrates. This was part of a reneweddescription of paroxetine hydrochloride hydrate form II, which has previously been determined as ananhydrate. However, new research, including PXRD measurements performed at variable humidity,has documented the hypothesis that paroxetine hydrochloride hydrate form II is a non-stoichiometrichydrate. This particular example points out the difficulty connected with differentiation betweenvarious inner packing systems of water molecules in hydrates, which represents a challenge for bothscience and industry. An interesting case is the relation between one of the quinaldine hydrochlorideanhydrate (form B) and substance’s dihydrate [87]. Characteristic of stoichiometric hydrates, hysteresisbetween sorption and desorption isotherms is observed. Nonetheless, it is very small, which indicateshigh structural closeness of hydrate and anhydrate as well as the fact that this reversible phasetransformation has a low energy barrier. This agrees with other experiments indicating that dihydrateis the most kinetically stable form of quinaldine hydrochloride. Once more, such data play an importantrole in pharmaceuticals’ manufacturing and, furthermore, in their storage.

An important aspect of the (de)hydration kinetics is the number of the reaction steps. It raises thetopic of possible metastable intermediate phases. There are a fair amount of examples. At 95% RH and25 ◦C, acyclovir anhydrate form II transforms firstly into hydrate 3:2 (form V) and afterwards, underthe same conditions, into dihydrate (form VI) [120]. Another example is ondansetron hydrochloridedihydrate. After significant structural rearrangement is that it changes in the first step into hemihydrateand later on into anhydrate [144]. In the first step, the hydrogen bonding is altered. The second stageimplies structural differences as well. This time, a void is formed and the volume of the hydrophobiclayer is changed. In contrast, a two-step and quite atypical (at first, loss of 1–1.5 water molecules,and later, loss of 2.0–2.5 water molecules) dehydration of catechin 4.5-hydrate triggers no structuralchanges [145]. The reactions are reversible. For the most precise determination of this very specific

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process, both experimental (13C CPMAS NMR, 1H NMR) and calculation (GIPAW-NMR, CSP) methodshave been applied.

Another factor that influences the dehydration mechanism is the material preparation process.Non-micronized potential dyslipidemia reducing agent, non-stoichiometric dihydrate, follows atwo-step reaction, whereas its micronized form undergoes a one-step dehydration [124]. The twoprocesses differ distinctly. For their description, a combination of TGA, DSC, PXRD, SXRD, 15N ssNMR,and DFT has been applied. All the above mentioned examples show how diverse multi-step reactionsof (de)hydration are and, at the same time, indicate the need to employ a variety of research techniques.

5. Hydrates’ Stability, Selected Cases within the Last 10 Years

Kinetic stability could be briefly defined as a reaction’s probability to occur in the given conditions.However, a kinetically stable compound is often at the same time thermodynamically metastableas it has not reached a global energy minimum because of the high energy barrier of a phasetransition [81]. Nevertheless, application of different external conditions could influence the directionof the hydrate-anhydrate reaction. The practical importance of this subject explains numerous scientificarticles published on the topic.

The stability order of hydrates/anhydrates is a function of RH, temperature, and aw. [87,116,132,146].To name an example, gabapentin anhydrate is more stable at lower water fractions and risingtemperatures when compared with the drug’s hydrate, whereas gabapentin monohydrate is favorableat higher water fractions and lower temperatures [129]. Such behaviour concerning anhydrate beingmore stable at lower RH and a hydrate at higher RH is a general rule. However, at very high RH, drugsubstances are prone to degradation and this is the reason the ICH guidelines suggest performingstability studies in accelerated conditions of 40 ◦C/75% RH [147]. Though, there are some exceptions,like levothyroxine sodium pentahydrate which remains stable even at high RH rates and is unstable atlower ones [148]. The explanation is most probably the presence of unusually high number of watermolecules within this hydrate.

Kinetic stability is also an aspect that attracts attention while investigating hydrate co-crystalsbecause it is connected with solubility, which in turn determines bioavailability [149–153]. One of theexperiments showed that kinetically stable imatinib hydrate, after associating syringic acid, formsa metastable co-crystal with higher permeability through membranes [154]. Similar results deliverexamination of griseofulvin-acesulfame hydrate. It presents remarkable thermal stability at differentRH up to 13 weeks and has an improved dissolution rate when compared with the griseofulvinhydrate [155].

Stability is a decisive factor for storage aspects [156,157]. It depends on the material’s purity [86,158].An addition of other phase can induce nucleation of another form and, as a consequence, a polymorphictransformation could take place [158]. Later on, it could influence the process of formulation preparationas well. One of the first stages of drug industrial production is grinding, which can induce polymorphicchanges and (de)hydration [159]. For this reason, studies including this process are performed. Forexample, neomycin sulphate hydrate lost its water after 2.5 h of grinding, but a hydrate could berestored after storage in 71% and 99% RH [157]. Conversely, theophylline and caffeine anhydratestransformed into hydrates after 5 min long grinding with water [160].

The next step in the drug industrial production is often wet granulation. As the storage stage,it represents a potential risk of unwanted hydration. For example, in the case of theophylline, wetgranulation shows a very specific impact on the tableting process; that is, at 40 ◦C, theophyllineanhydrate is subjected to the following transitions: THA→THM→THA, where THA is theophyllineanhydrate and THM is theophylline monohydrate. However, when wet granulation is performed at60 ◦C, the compound stays in a form of THA the whole time [161]. Irrespective of the fact that, in theend, in both cases, THA is maintained, the differences in tablet hardness, disintegration time, anddissolution rate, assigned to the hydration changes during granulation, are detectable [161].

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6. Influence of Excipients on API Hydrates

Excipients can influence APIs’ nucleation or their solubility [108]. Investigation of the wholeformulation instead of merely the active ingredient poses a challenge in the pharmaceutical analysisfor each type of API. In API hydrates, it is probably even more pronounced owing to the presenceof water in APIs’ crystal lattice [77,119,123]. The majority of cases reported on the excipients’ rolein a drug formulation where API has a form of a hydrate concern polymers. Their direct influenceon the (de)hydration process has been analyzed in detail. For instance, a study on sodium naproxenhydrate indicates that, in tablet pressing conditions, polivinylpyrrolidone (PVP) delays, whereasmicrocrystalline cellulose (MC) facilitates, transformation into the anhydrate [137].

However, because, in most of cases, polymers prevent an opposite reaction, namely hydration,most studies concentrate on this aspect while examining formulations that include hydrated API.The polymers’ effect depends on both the manufacturing process parameters and polymers’ type. Forexample, inhibition of the caffeine hydrate formation by PAA (polyacrylic acid) is dependent on pHvalues. The highest process intensity is observed at low pH values. Moreover, the same study showsthat PVA (polyvinyl alcohol) with a higher molecular weight inhibits caffeine dehydration more thanPVA with shorter chains [109]. Similar results are delivered in the study on olanzapine. PVP K30,even if added at a very low concentration (0.002%) to the formulation during the wet granulationprocess, effectively prevents formation of olanzapine hydrate [162]. Another study indicates that potentinhibitors of olanzapine hydrate-formation are PVP and HPC (hydroxypropyl cellulose) [163,164].Interestingly, PEG (polyethylene glycol) stimulates a further process up to higher hydrates. Anotherexample is the case of carbamazepine, whose hydration was inhibited by short chain substitutedcellulose like HPMC (hydroxypropylmethyl cellulose) and MC. The same trend has been observed forPVA and PVP; that is, longer chains exhibited lower efficiency [155].

Water originating from excipients can alter the solubility rate of API, and thus influence the drug’sbioavailability [62]. Especially excipients with high water-absorbing potential, like MC or HPC, candelay the start of a hydrate formation in an aqueous environment (e.g., wet granulation), as has beenreported for piroxicam [116]. Nevertheless, sometimes, excipients also cause a stability decrease informulations containing API hydrates [128].

A wide range of excipients’ impact on the final drug product justifies the importance of researchon this topic. The complexity of the problem requires application of different analytical techniques.To determine the structural purity, Near Infrared (NIR) is used because, unlike the thermal methods, thistechnique may overcome interference from excipients [120]. To observe anhydrate–hydrate changes,mostly Raman spectroscopy is applied [62,109,161], whereas explanation of excipients’ protectiveeffect on anhydrate APIs is provided by PXRD and NMR measurements [55,77]. This hydrationprevention is determined by dense H-bonding network emerging between API and polymers andby the hydrophobic layer formed by excipients around the active ingredient. These factors preventwater molecules from coming into contact with the host substance and, as a result, no hydrate in drugformulation is observed.

7. Analysis of Commercial Solid Dosage Forms

Solid dosage forms are the most popular methods of drug delivery due to their numerousadvantages like convenience of administration and facility of mass production. However, solid statepharmaceutical analysis, suffers from some inconveniences. For example, the inability to separatethe components of the homogenous samples such as solid drug formulations and the low methodsensitivity when compared with the solution methods. However, only the solid state analysis canprovide information on the solvated forms of APIs used in the formulation. Though all of the analyticalmethods described in the first section of this review (PXRD, FT-IR, Raman, ssNMR, and DSC), withthe exception of SCXRD, can possibly be used in the analysis of solid dosage form, and each of themhave their advantages and disadvantages. When choosing the appropriate one in a particular case,the attention should be paid to the qualitative and quantitative composition of the excipients in the

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solid dosage form, which may interrupt the analysis, as well as the API content. In a recent study [38],the performance of selected solid-state analytical methods (PXRD, FT-IR, and ssNMR) in the analysisof the solid drug forms with low concentration of an active ingredient has been compared. The authorsfound that the only method that unambiguously confirmed the presence of a particular solvated formof API in the studied drug was ssNMR (Figure 7). Because of the possibility of modification of pulsesequence and the manipulation in the ssNMR experimental registration parameters like recycle delay,inversion recovery CP evolution time, and CP contact time, it was possible to observe the signals of thechosen components of the drug formulation. The other studied methods (PXRD, FT-IR) have failedbecause of the overlapping of the excipients’ signals with the signals of an API.

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time, it was possible to observe the signals of the chosen components of the drug formulation. The other studied methods (PXRD, FT-IR) have failed because of the overlapping of the excipients’ signals with the signals of an API.

Figure 7. Estrofem mite ® (3), 17-β-estradiol hemihydrate (2), and anhydrous 17-β-estradiol (1) 13C CP MAS NMR spectra. Solid state NMR analysis enabled to determine which form of 17-β-estradiol is present in the commercial dosage form, despite the low content of active pharmaceutical ingredient (API) (<5%). Source: author’s archive, more details in [38].

8. Conclusions

Polymorphism is a phenomenon of key importance for the pharmaceutical industry. Likewise, the possibility of solid APIs to exist at different levels of hydration represents another way to improve the APIs’ properties by altering their solid state structures. However, differences in the level of hydration can affect APIs’ physical and chemical properties, such as solubility, dissolution rate, melting point, stability, and tabletability. For this reason, a thorough screening for differently hydrated forms of APIs, as well as determination of their structures together with the mechanism and kinetics of (de)hydration, are necessary in the modern drug production process.

Among the instrumental and computational techniques presented in this review, there is none that, used alone, would answer all the questions regarding the above-mentioned structural issues. All of the described analytical methods are complementary to each other. Only when used as a set, they allow precise determination of APIs’ structure and stability. Moreover, in this way, they provide a tool to track structural changes that occur during the production process as a result of different humidity, temperature, or pressure conditions.

Author Contributions: Conceptualization, E.J., A.H.M., Ł.S., D.M.P. and M.Z.-P.; investigation, E.J., A.H.M., Ł.S., D.M.P. and M.Z.-P.; writing—original draft preparation, E.J., A.H.M. and Ł.S.; writing—review and editing, A.H.M., Ł.S. and D.M.P.; supervision, A.H.M. and Ł.S.; project administration, Ł.S. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Figure 7. Estrofem mite ® (3), 17-β-estradiol hemihydrate (2), and anhydrous 17-β-estradiol (1) 13C CPMAS NMR spectra. Solid state NMR analysis enabled to determine which form of 17-β-estradiol ispresent in the commercial dosage form, despite the low content of active pharmaceutical ingredient(API) (<5%). Source: author’s archive, more details in [38].

8. Conclusions

Polymorphism is a phenomenon of key importance for the pharmaceutical industry. Likewise, thepossibility of solid APIs to exist at different levels of hydration represents another way to improve theAPIs’ properties by altering their solid state structures. However, differences in the level of hydrationcan affect APIs’ physical and chemical properties, such as solubility, dissolution rate, melting point,stability, and tabletability. For this reason, a thorough screening for differently hydrated forms of APIs,as well as determination of their structures together with the mechanism and kinetics of (de)hydration,are necessary in the modern drug production process.

Among the instrumental and computational techniques presented in this review, there is nonethat, used alone, would answer all the questions regarding the above-mentioned structural issues.All of the described analytical methods are complementary to each other. Only when used as a set, theyallow precise determination of APIs’ structure and stability. Moreover, in this way, they provide a tool

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to track structural changes that occur during the production process as a result of different humidity,temperature, or pressure conditions.

Author Contributions: Conceptualization, E.J., A.H.M., Ł.S., D.M.P. and M.Z.-P.; investigation, E.J., A.H.M.,Ł.S., D.M.P. and M.Z.-P.; writing—original draft preparation, E.J., A.H.M. and Ł.S.; writing—review and editing,A.H.M., Ł.S. and D.M.P.; supervision, A.H.M. and Ł.S.; project administration, Ł.S. All authors have read andagreed to the published version of the manuscript.

Funding: This research received no external funding.

Conflicts of Interest: The authors declare no conflict of interest.

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